The invention relates to a method for fractionating various components contained in a sample solution by using a liquid chromatograph mass spectrometer.
Heretofore, there has been known a fraction chromatograph wherein a plurality of components contained in a sample is separated and collected by using a chromatograph device, such as high performance liquid chromatograph (hereinafter referred to as “HPLC”).
FIG. 1 is a block diagram for showing an example of a structure of a fraction chromatograph using HPLC. An eluant, i.e. mobile phase, stored in an eluant tank 1 is sucked by a pump 2 and is transferred to flow into a column 4 through a sample introduction portion 3 at a predetermined flow rate. A sample solution injected into the mobile phase at the sample introduction portion 3 is introduced into the column 4 together with the mobile phase, and while passing through the column 4, components in the sample solution are separated and eluted.
A detector 5 detects the components eluted from the column 4 sequentially and sends detection signals to a signal process portion 6. All or a part of the eluate passing through the detector 5 is introduced into a fraction collector 8. The signal process portion 6 prepares a chromatogram based on the detection signals obtained from the detector 5, and a control portion 7 provides the fraction collector 8 with a control signal for fractionation based on a peak appearing on the chromatogram at real time. The fraction collector 8 controls an electromagnetic valve and the like based on the control signal, and distributes the eluate to vials corresponding to the respective components.
Recently, there has been widely used a liquid chromatograph mass spectrometer (hereinafter referred to as “LC/MS”) using a mass spectrometer (hereinafter referred to as “MS”) as a detector of HPLC. In the MS, various components contained in an introduced sample are separated and detected according to a mass number, i.e. mass/charge. Therefore, even if a plurality of components is overlapped at the same elution time, it is possible to separate these components for a qualitative analysis and a quantitative analysis.
FIG. 2 is a view showing an example of a configuration of the LC/MS apparatus. In the LC/MS, a mass scan is carried out within a certain mass range. Then, an intensity of an ion separated in every mass number is detected sequentially, and a relationship between the mass number and the intensity is created to obtain a mass spectrum. Also, a total ion chromatogram (hereinafter referred to simply as “chromatogram”) can be obtained by repeatedly carrying out the mass scan, integrating the ion intensity in every scan regardless of the mass number and examining a change of the total ion intensity with time. Further, by focusing on a specific mass number, a mass chromatogram can be obtained by examining a change of the ion intensity having the mass number with time for every scan.
In general, in the LC/MS, a sample is ionized with a soft ionization method (an electro spray method, an atmospheric-pressure chemical ionization method and the like). Accordingly, different from an EI (electron impact) ionization method used in the GC/MS, it is possible to obtain a simple chromatogram in which only such ions as [M+H]+ and [M+Na]+, which are produced through an addition of a proton or a salt in the solvent to a material, are detected (hereinafter, called “molecular ion detection mode”). When it is necessary to obtain information regarding a molecular structure, a method called CID (collision induced dissociation) is used. In this method, a molecular ion is produced in a nebulizing chamber 11. Then, a voltage different from an ordinary voltage is applied to an electrode disposed in an immediate chamber 15 to induce the CID to create a fragment of the molecular ion, thereby detecting the fragment (hereinafter, called “fragment ion detection mode”).
When the LC/MS is used for the fraction chromatograph, it is necessary to determine a timing of fraction based on chromatogram data for preparing a chromatogram or mass-chromatogram. Normally, the chromatogram data is calculated from a number of mass spectrum data obtained by a single mass scan according to a predetermined process condition set (for example, a sum of intensities of ions having a specific mass number, a sum of intensities of ions within a predetermined mass range and the like). Therefore, a single chromatogram data is obtained per a single mass scan.
When a mass scan is alternately switched between the molecular ion detection mode and the fragment ion detection mode within a wide mass range (for example, m/z: 20-2000) for the analysis, a chromatogram data obtained at a certain time point t is a value calculated based on the mass spectrum obtained in the molecular ion detection mode, and the subsequent chromatogram datum obtained at t+Δt is a value calculated based on the mass spectrum obtained in the fragment ion detection mode.
Generally, the background noises are at different levels in the molecular ion detection mode and the fragment ion detection mode. As a result, as shown in FIG. 7(a), a chromatogram based on a mass spectrum obtained in the molecular ion detection mode has a base line different from that of a chromatogram based on a mass spectrum obtained in the fragment ion detection mode. Therefore, as shown in FIG. 7(b), when the chromatogram data obtained whenever the mass scan is switched between the two measurement modes is continuously combined, a resultant chromatogram curve has a saw teeth shape.
Also, when a measurement is performed in a SIM mode in which only a predetermined mass number is continuously monitored to obtain a chromatogram, each chromatogram exhibits a chart like, for example, one shown in FIG. 8(a). As a result, as shown in FIG. 8(b), when the chromatogram data is continuously combined, a resultant chromatogram curve has a saw teeth shape.
In either case, it is difficult for the control portion 7 to accurately determine a starting point and a terminal point of a peak from such chromatogram data. Therefore, it is impossible to determine when each component should be fractionated, or an erroneous control signal is sent to the fraction collector 8. For this reason, when the conventional LC/MS is used for the fractionation operation, it is difficult to fractionate while alternately switching the measurement modes, and it is necessary to perform the fractionation operation in a single measurement mode, resulting in poor operational efficiency.
In view of the above problems, the present invention has been made, and an object of the present invention is to provide a liquid chromatograph mass spectrometer for obtaining a chromatogram to operate a fraction collector normally even when a mass spectrometry analysis is carried out while switching measurement conditions such as the molecular ion detection mode and the fragment ion detection mode. As a result, it is possible to complete a proper fractionation operation only through a single analysis.
Further objects and advantages of the invention will be apparent from the following description of the invention.